Elevated NS1 serum levels reduce CD119 expression and CXCL-10 synthesis in patients with dengue hemorrhagic fever

ABSTRACT Background: The intensity of dengue virus (DV) replication and circulating non-structural protein 1 (NS1) levels may promote changes in the human immune response and favor severe forms of infection. We investigated the correlations between NS1 with CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels, and IFN-γ receptor α chain (CD119) expression, and CXCL10 production by peripheral blood mononuclear cells (PBMCs) stimulated with recombinant IFN-γ in DV-infected patients with different clinical forms. Methods: Dengue virus NS1, CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels were measured in 152 DV-infected patients with different clinical forms and 20 non-infected individuals (NI) using enzyme-linked immunosorbent assay (ELISA). In addition, we investigated the CXCL-10 production after in vitro IFN-γ stimulation of PBMCs from 48 DV-infected individuals (with different clinical forms of dengue fever) and 20 NI individuals using ELISA, and CD119 expression on CD14+ cells with flow cytometry. Results: Patients with dengue hemorrhagic fever (DHF) had significantly higher NS1, CXCL-8, and CXCL-10 serum levels than those with classic dengue fever (DF). The response of PBMCs to IFN-γ stimulation was lower in patients with DHF than in those with DF or dengue with complications (DWC), with lower CD119 expression and reduced CXCL-10 synthesis. In addition, these alterations are associated with high NS1 serum levels. Conclusions: Patients with DHF reported high NS1 levels, low CD119 expression, and low CXCL-10 synthesis in PBMCs, which may be associated with infection progression and severity.


INTRODUCTION
Dengue virus (DV), transmitted by the female Aedes aegypti mosquito, infects an estimated 100 to 400 million individuals worldwide annually 1,2 .It is endemic to Africa, the Americas, Southeast Asia, the Eastern Mediterranean, and the Western Pacific.In 2022, significant dengue fever outbreaks occurred globally, particularly in the Americas, where nearly three million cases were reported.Brazil has accounted for more than 2.3 million cases and reported 1,016 deaths 3 .
Four serotypes of DV exist: DV-1, DV-2, DV-3, and DV-4 2,4,5 .It is a single-stranded RNA virus that synthesizes a polyprotein that produces three structural (C, prM, and E) and seven non- structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).The latter are involved in viral RNA replication 2,6,7 .NS1 is a viral glycoprotein occurring at different levels in the serum during the acute phase of infection and serves as a marker for early diagnosis of DF 8,9 .Furthermore, it could be associated with the etiopathogenesis of this disease 9,10 .
The immune response to DV, although capable of containing infection and promoting recovery, may contribute to the development of more severe forms of the disease 10,11,12 .IFN-γ plays a central role in controlling viral replication and enhancing resistance to infection [13][14][15][16][17][18][19] and the regulation of IFN-γ receptor expression on the cell surface could serve as a mechanism through which cells modify their response to IFN-γ 19,20 .DV-infected individuals exhibit elevated CXCL-10 (IP-10) levels associated with the febrile period observed during the acute phase 21,22,23 ; however, studies have not yet investigated the association between NS1 serum levels, inflammatory mediators, cellular response to IFN-γ and the development of different clinical forms of DF.Therefore, we hypothesized that NS1 levels could interfere with IFN-γ-induced CXCL-10 synthesis and production of inflammatory mediators during an immune response to DV in humans.
Elevated CXCL-8 (IL-8) levels have been linked to plasma leakage in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).Individuals with thrombocytopenia and DV infections exhibit significantly higher CXCL-8 levels [23][24][25][26][27][28][29] .One study using an in vitro model demonstrated that the synthesis of high levels of CXCL-8 increased the risk of developing severe forms of the disease, particularly secondary infections 30 .However, the precise relationship between NS1 levels and changes in the innate immune response remains unclear.Thus, we assessed the circulating NS1 levels and their correlation with CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels.In addition, in vitro CXCL-10 production by peripheral blood mononuclear cells (PBMCs) in response to IFN-γ stimulation and IFN-γ receptor expression was evaluated in patients with different clinical forms of DF.

Study group and case classification:
A total of 213 patients were initially evaluated for participation by a team of infectious disease specialists at a dengue outpatient clinic.Of these, 51 were excluded from the study (24 did not have a positive laboratory diagnosis of DV infection, 1 had a positive serology for HIV, and 26 had incomplete material for analyses or inconclusive clinical and laboratory data).Therefore, the final group of patients diagnosed with DF comprised 152 patients (74 females and 78 males).Among them, 104 had samples collected only for serum, whereas the remaining 48 had samples collected for serum and PBMCs.Patients who sought care for suspected DV infection between the 2nd and 5th days of fever onset and who met the criteria were recruited into the study.Patients were followed and treated at the Dengue Outpatient Clinic of the Federal University of Triângulo Mineiro, Abadia Emergency Unit, and São Marcos Hospital between 2011 and 2014.Patients were classified after the analysis of all clinical data and laboratory results (obtained from at least two sample collections, one in the acute phase and the other in convalescence) by infectious disease specialists.A total of 106 DV-positive patients were classified as having classical DF, 31 as having dengue complications (DWC), and 15 as having DHF.Dengue cases were classified as DF or DHF according to the guidelines proposed by the World Health Organization (WHO) 31 .We applied the traditional classification because of the specific characteristics of our experimental work to test our hypothesis (evaluate whether elevated NS1 serum levels influence parameters specifically associated with the definition of DHF, such as platelet count, hematocrit, and others associated with immunopathogenesis).In addition, the 2009 WHO guidelines are more directly focused on clinical management and not broadly used in research 32 .For the control group, 20 healthy individuals (10 females and 10 males) without a current or recent history of fever or any other disease symptoms, who were not infected with DV, tested negative for NS1 and anti-DV IgM/IgG antibodies, and had not been vaccinated against yellow fever, were recruited and included as non-infected healthy controls (NI).Samples were obtained during the acute phase (days 2-5 after symptom onset) and at the beginning of the convalescent phase (days 9-12 after symptom onset).In addition, laboratory data (hemogram, NS1 levels, and anti-DV IgM/IgG), signs and symptoms (fever, headache, loop test, retro-orbital pain, diarrhea, rash, prostration, nausea, vomiting, epistaxis, gingivorrhagia, hematuria, petechiae, metrorrhagia, gastrointestinal bleeding, myalgia, arthralgia, severe abdominal pain, and painful hepatomegaly), and outcomes (hospitalization, death, and complications) were collected.Data on sex, age, and case classifications are presented in Table 1 Blood samples collection and processing: During the acute phase, 5 mL of blood without anticoagulants was collected from 152 DV-infected individuals and 20 NI healthy controls to obtain serum samples.In addition, 20 mL of heparinized peripheral venous blood was collected during the acute phase from 48 DV-infected patients and 20 NI healthy individuals for PBMC isolation.During the convalescent phase, 5 mL of blood without anticoagulants was collected from 152 DV-infected individuals to obtain serum samples.All serum samples, collected without anticoagulant, were centrifuged at 500 ×g for 10 min at 21 o C and stored in aliquots at −86°C until further use.

Dengue fever serological diagnosis:
Dengue feverspecific IgM and IgG were detected in serum samples using the Panbio TM dengue-specific IgM and IgG capture enzyme-linked immunosorbent assay (ELISA) kit (Abbott Laboratories Inc., Abbott Park, IL, USA) according to the manufacturer's instructions.The presence of DV NS1 antigen was determined using Platelia™ Dengue NS1 Ag immunoenzymatic assay (Bio-Rad Laboratories Inc., Marnes-la-Coquette, France) using the qualitative protocol performed according to the manufacturer's instructions.
Serum soluble NS1 levels: NS1 serum levels were measured using a quantitative protocol of Platelia™ Dengue NS1 Ag enzyme immunoassay (Bio-Rad Laboratories, Marnes-la-Coquette, France) according to the manufacturer's instructions.The quantitative protocol (Bio-Rad technical support version 05/2008 of Platelia Dengue NS1 Ag: quantitative detection of DV NS1 antigen in human serum or plasma by enzyme immunoassay) used a different conjugate dilution (1:5000) and a specific formula to calculate NS1 units based on human recombinant NS1 (positive control) and the calibrators of the kit.NS1 serum levels were calculated by multiplying the OD of the sample tested using the 1:5000 diluted conjugate by 150 as follows: Sample NS1 (BRU/mL) = Sample OD × 150/R4m, where R4m is the mean OD value obtained from cut-off control duplicates.This protocol was used for all samples confirmed positive using the qualitative protocol, and the results are expressed in Bio-Rad units per milliliter (BRU/mL).
Cytokine and chemokine quantification: IFN-γ, IL-12p40, CXCL-8, and CXCL-10 concentrations in serum and 18 h cell culture supernatants were measured using ELISA kits with monoclonal antibody pairs according to the manufacturer's specifications (BD Biosciences, San Jose, CA, USA).The absorbance was determined using a Modulus microplate reader (Promega, Madison, WI, USA), and the absorbance at 450 nm was subtracted from that at 570 nm.Cytokine/chemokine concentrations were calculated and the results are expressed in pg/mL.

Cytometry analysis strategy:
The CD119 (IFN-γR1) expression on CD14 + PBMCs was obtained by drawing a gate on FL1 (CD14 + FITC fluorescence) versus the SSC parameter.The CD14 + cells in region 1 (R1) were further analyzed in a PE fluorescence channel (FL2) representing CD119 expression.
Statistical analyses: Data were analyzed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA).The Mann-Whitney U test was used for comparisons between two groups, and the Kruskal-Wallis test was used for comparisons between three or more groups.Spearman's correlation coefficient (r) was calculated to determine the linear association between the two variables.The significance level was set at 5%.

Ethics statement:
The study protocol was approved by the Ethics Committee of the Federal University of Triângulo Mineiro, Uberaba, Minas Gerais State, Brazil (protocol n o .851).All participants provided written informed consent.

• Circulating NS1 levels during the acute phase and convalescence
The NS1 antigen was detected in the circulation of DVpositive patients from the acute phase to the recovery period, with significantly higher viral protein levels observed in the acute phase (p-value < 0.01) than in the initial convalescent period after infection (Figure 1A).During the acute phase, NS1 serum levels varied significantly among individuals, ranging from 0.2 × 10 1 to 6.0 × 10 5 BRU/mL (Figure 1B).Patients with DHF and DWC exhibited higher NS1 levels than those with DF (p-value < 0.01) (Figure 1C).
The NS1 serum levels showed a heterogeneous distribution among DV-positive patients (median = 8.4 × 10 3 BRU/mL) during the acute phase (Figure 1B), and two groups were established: patients with low NS1 serum levels (NS1 levels < median) and those with high NS1 serum levels (NS1 levels ≥ median).Individuals with high NS1 serum levels had lower platelet count/μL (p-value < 0.01) than those with reduced NS1 serum levels (Figure 1D).A negative correlation was observed during the acute phase (Figure 1E and 1F) between platelet counts and circulating NS1 levels, especially in the high NS1 patient group (Figure 1F, r = −0.5008).
At the beginning of the convalescence period (day 9), as shown in Figure 2E, patients with DHF exhibited a sustained elevation in CXCL-8 levels compared to DF and NI individuals (p-value < 0.01).

• IFN-γ receptor alpha chain expression in CD14 + cells
We investigated whether the reduced cellular response to IFN-γ in DHF was associated with a modulation of the IFN-γ receptor α chain expression (CD119 or IFN-γR1) in CD14 + cells (Figure 4D and 4E).
As shown in Figure 4D, during the acute phase, CD14 + cells in patients with DF exhibited higher CD119 expression than those in patients with DHF (p-value < 0.01), DWC (p-value < 0.01), or NI individuals (p-value < 0.01).CD14 + cells from patients with higher NS1 serum levels exhibited lower CD119 expression (p-value < 0.01) than those from patients with lower NS1 concentrations (Figure 4E).

DISCUSSION
NS1 serum concentrations displayed significant heterogeneity during the acute phase of DV infection, with patients having higher NS1 levels than those with DF.Thus, individuals with DF may have a more effective immune response during the acute phase, facilitating DV clearance and the subsequent reduction in soluble viral antigens throughout infection.High NS1 serum levels may contribute to the development of severe hemorrhagic forms such as DHF because NS1 may induce abundant anti-NS1 antibodies that cross-react with platelets, causing thrombocytopenia and altered coagulation [8][9][10]12,33 . Withi this context, our findings are consistent with those of previous studies in which individuals with DHF exhibited elevated NS1 serum levels compared to those with DF 9,10,12,34 .The synthesis of anti-NS1 antibodies can lead to cross-reactivity against epitopes present in platelets and vascular endothelium, inducing apoptosis of endothelial cells, platelet aggregation, and complement system-mediated lysis.In addition, these processes could contribute to hemorrhage and plasma leakage during DV infection [35][36][37] .
We observed that infected individuals with increased NS1 levels exhibited reduced platelet counts, as reported previously [33][34] .The literature reports that anti-DV NS1 antibodies bind to protein disulfide isomerase on the platelet surface to inactivate this protein and αIIbβ2 integrin, which inhibits platelet adhesion and aggregation [35][36][37][38] .This process also promotes increased phagocytosis due to the opsonization of platelets 39 .Although endothelial dysfunction is complex and poorly understood, our findings suggest that high NS1 serum levels detected during the acute phase of infection could be associated with the development of severe forms, such as DHF/DSS.Our study demonstrated that patients who developed severe forms, such as DHF, had extremely high serum levels of NS1 during the acute phase.High concentrations of NS1 can produce abundant anti-NS1 antibodies that can cross-react with epitopes present in endothelial cells, causing temporary dysfunction of the endothelium [35][36][37][38][39] Furthermore, it can bind to platelets, causing numerous alterations such as opsonization, agglutination, and microthrombi formation [37][38][39] .Thus, these factors may be associated with the development of severe forms, such as DHF/DSS, during the beginning of the defervescence phase of DV infection.However, further studies are required to describe the mechanisms involved in detail and reinforce these hypotheses.
Studies have highlighted increased IFN-γ and IL-12 levels in DV infections across different clinical forms 15,16,45,46 .Individuals with DF maintain elevated serum IFN-γ and IL-12 levels, which promote infection 15,16,17,45,46 .However, individuals with DHF experience peaks of IFN-γ (mainly before plasma leakage) 17 and IL-12 46 , followed by reduced serum levels.Individuals with high NS1 levels exhibited elevated CXCL-8 and CXCL-10 levels, but not IFN-γ and IL-12p40 levels.The concentrations of both chemokines increased proportionally with the NS1 levels, indicating that NS1 may affect certain parameters of the antiviral response during acute infection.Increases in CXCL-8 synthesis, vascular permeability, and plasma leakage are possibly triggered by systemic alterations 40,47 , and anti-DV NS1 antibodies promote CXCL-8 synthesis after endothelial activation 48 .Therefore, high levels of CXCL-10, CXCL-8, and NS1 may be associated with excessive and unregulated inflammatory responses during DV infection.
We demonstrated that PBMCs from individuals with DHF exhibited a diminished response to IFN-γ, as indicated by a decreased CXCL-10 synthesis compared to individuals with DF and DWC.Similarly, individuals with high NS1 levels exhibited a poor cellular response to IFN-γ, resulting in no increase in CXCL-10 synthesis after in vitro stimulation.In contrast, patients with reduced NS1 levels demonstrated increased IFN-γ response.We demonstrated that DV suppresses cellular immunity during the peak viral replication by temporarily reducing IFN-γ synthesis by PBMCs, probably affecting the stability and activity of the IFN-γ/IFN-γ receptor system 18 .Dendritic cells (DC) infected in vitro with DV produce less CXCL10 than non-infected DCs, and the DC maturational state is modified by the presence of DV 49 .This affects the etiopathogenesis of the disease because the reduced capacity of DV-infected cells to stimulate CD4 + T lymphocytes impacts the efficiency of the immune response, causing reduced control of the viral load 49 .The evasion mechanisms employed by DV may affect the IFN-mediated antiviral response.Specifically, they hinder the phosphorylation of Tyk2 tyrosine kinase (a STAT activating molecule) 50 , and the viral protease NS2B3 acts by reducing IFN-γ synthesis 51 .Therefore, the observed lower response to the IFN-γ stimulus in PBMCs from individuals with DHF suggests a modulation of the IFN-γ/IFN-γ receptor system, possibly associated with high NS1 concentrations, impairment of viral replication control mechanisms, and modulation of inflammatory response pathways.Our findings support the hypothesis that changes induced by high levels of NS1 affect the IFN-γ signaling pathway, and unregulated signaling may also lead to severe inflammation 52 .
In addition to increased NS1 serum levels and a reduced cellular response to IFN-γ detected in DHF patients, reduced IFN-γ receptor CD119 expression was observed in CD14 + cells in these patients.This reduction was associated with a higher concentration of circulating NS1 because of a lower expression of CD119 in CD14 + monocytes in the high NS1 patient group.Although the involvement of NS1 in viral RNA replication has been recognized, its precise function in DF pathogenesis remains incompletely Garcia FG et al. • NS1 levels and CXCL-10 synthesis in dengue fever elusive 8,53,54,55 .Further investigations are required to explore the potential mechanisms by which CXCL-8, a powerful vasoactive chemokine, alters the immune response.Moreover, increased NO levels produced during the inflammatory response may interfere with CD119 expression in infected cells 56 .Our results suggest a DV escape mechanism subverts the host immune response, particularly the cellular response to IFN-γ stimulation and CXCL-10 synthesis.
Among the limitations of our study is the non-detection of the predominant virus serotype, although the identification of specific circulating DV serotypes was not the main concern of our study.During this epidemic, official agencies from our state and municipality analyzed only a small percentage of samples from our city, and DV serotype 1 was the most frequently found in our region.We simultaneously serotyped a few of the 152 samples included in the study (n = 8) and detected serotype 1 in all analyzed patients.In addition, the panel of cytokines and chemokines investigated could have been broader.However, considering numerous biological samples analyzed in this study, we focused on key mediators associated with NS1 levels, course of infection, and development of DHF.In contrast, our work opens perspectives for investigating the molecular mechanisms involved in the likely suppression of the cellular response to IFN-γ by NS1 during the critical period of the acute phase and defervescence.Another open perspective is to investigate the possible function of anti-NS1 antibodies in the immunopathogenesis of dengue.
In conclusion, individuals with severe forms of DF exhibit immune response alterations during the acute phase, including increased serum NS1 levels that are associated with platelet reduction, higher concentrations of CXCL-8 and CXCL-10, and reduced IFN-γ levels.Lower expression of CD119 in CD14 + monocytes detected in DHF patients when compared to DF patients, is related to a diminished cellular response to IFN-γ causing reduced CXCL-10 synthesis.Therefore, the combination of alterations detected may contribute to the progression and severity of DF.

FIGURE 1 :
FIGURE 1: Serum concentration distribution of dengue virus non-structural protein 1 (NS1) and platelet number in infected individuals (n = 152).(A) NS1 serum levels in acute febrile phase (days 2 to 4) and beginning of convalescence (day 9).(B) NS1 circulating serum levels during the acute phase, where the line represents the median of 152 DV-infected individuals.(C) NS1 serum levels in the acute febrile phase in individuals with dengue fever (DF), dengue with complications (DWC), and dengue hemorrhagic fever (DHF) in BRU/mL.(D) Platelet count/ µL in infected individuals with low (NS1 < 8.4 × 10 3 BRU/mL) and high NS1 serum levels (NS1 ≥ 8.4 × 10 3 BRU/mL) during the acute phase.(E) Spearman's correlation between NS1 levels and platelet count/µL in infected individuals from the low NS1 group during the acute phase.(F) Spearman's correlation between NS1 levels and platelet count/µL in infected individuals from the high NS1 group during the acute phase.The (*) symbol between the two groups indicates a statistically significant difference (p < 0.05).